Thiourea derivatives as chelating agents for bioconjugation of rhenium and technetium.

Potential tetradentate thiocarbamoylbenzamidine derivatives H4L have been synthesized from the corresponding benzimidoyl chlorides and triglycine. They are suitable chelating agents for the oxidotechnetium(v) and oxidorhenium(v) cores and form stable, neutral [MO(HL)] complexes with an equatorial SN3 coordination sphere and an additional, uncoordinated carboxylic group, which can be used for bioconjugation. Representatives of the rhenium and 99Tc products have been isolated and analyzed with spectroscopic methods and X-ray diffraction. Bioconjugates of these complexes with angiotensin-II have been synthesized and structurally characterized. Analogous 99mTc complexes have been produced and tested in vitro and in vivo. The experiments confirm a considerable stability for the [99mTc(HL)] product as well as for its bioconjugate and recommend this class of compounds for further bioconjugation studies towards clinical applications.

Combined use of Ovsynch and progesterone supplementation after artificial insemination in dairy cattle.

The objective of this study was to evaluate the effects of different Ovsynch protocols combined with progesterone (P4) supplementation after artificial insemination (AI) of Holstein-Friesian cows. Cows were randomly synchronized at 52 to 63 d after parturition with either the classical Ovsynch protocol (GnRH on d 0, PGF(2α) on d 7, GnRH 48 h after PGF(2α)) or with a modified Ovsynch protocol (second GnRH 60 h after PGF(2α)). On d 4 after timed AI (TAI), the cows were blocked by parity and randomly divided into 2 groups. Half of the cows were supplemented with P4 (P4+) by applying a P4-releasing intravaginal device intravaginally for 14 d, whereas the other half remained untreated (P4-). In 50% of randomly chosen cows, plasma P4 was measured on d 4, 5, and 18 after TAI. Sonographic pregnancy diagnosis was performed on d 33 after TAI in a total of 398 cows. Health status and body condition score (BCS) of all cows were examined at several stages of the study. Cows in the modified Ovsynch protocol tended to have higher P4 values on d 4 after TAI than cows in the classical Ovsynch protocol (2.1 ± 0.2 vs. 1.6 ± 0.2 ng/mL), but no difference in pregnancy per AI (P/AI) was observed between the 2 Ovsynch protocols (38.4% vs. 44.1%). Independent of the Ovsynch protocols, P4+ cows tended to have higher P/AI compared with P4- cows (44.4% vs. 38.1%). The retention of fetal membranes and BCS at the time of insemination affected P/AI. Moreover, an interaction between BCS at the time of insemination and P4 supplementation was apparent; that is, the difference in P/AI between P4+ and P4- cows was significant in cows with BCS ≥3.25. Progesterone-supplemented cows showed higher P4 values on d 5 (4.9 ± 0.2 vs. 2.6 ± 0.2) and d 18 (7.8 ± 0.2 vs. 6.3 ± 0.2) after TAI, respectively. In conclusion, the elongation of the time interval between the injections of PGF(2α) and the second GnRH from 48 to 60 h had no effect on P/AI. Progesterone supplementation after insemination improved the P/AI of the Ovsynch protocols, but this effect was more apparent in cows with BCS ≥3.25.

Expression of prostaglandin F2α (PGF2α) receptor and its isoforms in the bovine corpus luteum during the estrous cycle and PGF2α-induced luteolysis.

Prostaglandin F2α (PGF2α) induces luteolysis via a specific receptor, PTGFR. Although PTGFR mRNA expression in the bovine corpus luteum (CL) has been studied previously, changes in PTGFR protein and its localization are not fully understood during the life span of the CL. In addition to full-length PTGFR, several types of PTGFR isoforms, such as PTGFRα (type I) and PTGFRζ (type II), were reported in the bovine CL, suggesting isoform-specific luteal action. Full-length PTGFR mRNA in the bovine CL increased from the early to the mid-luteal phase and decreased during luteolysis, whereas PTGFR protein remained stable. PTGFR protein was localized to both luteal and endothelial cells and was expressed similarly during the life span of the CL. Like full-length PTGFR mRNA, PTGFRα and PTGFRζ mRNA also increased from the early to mid-luteal phases, and mRNA of PTGFRζ, but not PTGFRα, decreased in the regressing CL. During PGF2α-induced luteolysis, the mRNAs of full-length PTGFR, PTGFR,α and PTGFRζ decreased rapidly (from 5 or 15 min after PGF2α injection), but PTGFR protein decreased only 12 h later. Silencing full-length PTGFR using small interfering RNA prevented PGF2α-stimulated cyclooxygenase-2 (PTGS2) mRNA induction. By contrast, PGF2α could stimulate vascular endothelial growth factor A (VEGFA) mRNA even when full-length PTGFR was knocked down, thus suggesting that PGF2α may stimulate PTGS2 via full-length PTGFR, whereas VEGFA is stimulated via other PTGFR isoforms. Collectively, PTGFR protein was expressed continually in the bovine CL during the estrous cycle, implying that PGF2α could function throughout this period. Additionally, the bovine CL expresses different PTGFR isoforms, and thus PGF2α may have different effects when acting via full-length PTGFR or via PTGFR isoforms.

Low plasma progesterone concentrations are accompanied by reduced luteal blood flow and increased size of the dominant follicle in dairy cows.

To investigate the influence of low plasma progesterone (P(4)) concentrations on luteal and ovarian follicular development as well as endometrial gene expression in the concomitant and subsequent estrous cycle, 20 lactating dairy (Holstein Friesian and Brown Swiss x Holstein Friesian) cows received either a single treatment with 25 mg prostaglandin F(2α) (PGF(2α)) on Day 4 Hour 12 (PG1; n = 8), or two treatments (25 mg PGF(2α) each) on Day 4 Hours 0 and 12 (PG2; n = 12) of the estrous cycle (Day 1, Hour 0 = ovulation). In four cows, ovulation occurred between 4 and 6 d after the second PGF(2α) treatment; these cows and one lame cow were excluded. In the 15 remaining cows with physiological interovulatory intervals (18 to 24 d), P(4), luteal size (LS) and blood flow (LBF), as well as follicular size (FS) and blood flow (FBF), were determined daily until Day 4, immediately prior to (0 h) and 12 h after each PGF(2α) treatment, and then every 2 d, from Day 5 to 8 d after the subsequent ovulation. Because P(4) did not differ (P > 0.05) between PG1 and PG2, cows were regrouped according to their mean P(4) concentration from Days 7 to 15, either P(4) <2 ng/mL (P(4)L; n = 7) or P(4) >2 ng/mL (P(4)H; n = 8). In the treatment cycle, LS was smaller in P(4)L than P(4)H on Days 13 (P = 0.01) and 15 (P = 0.03), and LBF was lower in P(4)L than P(4)H on Day 15 (P = 0.02). The dominant follicle of the first follicular wave was larger in P(4)L than P(4)H on Days 13 (P = 0.03), 15 (P = 0.03), and 17 (P = 0.01). In the subsequent cycle, there were no significant differences between P(4)L and P(4)H for P(4), FS, LS, and LBF; however, FBF was lower (P = 0.01) in P(4)L than P(4)H on Day 7. In Group P(4)L, endometrial expressions of estrogen receptor α and oxytocin receptor were lower (P = 0.05 and P = 0.03, respectively) at the estrus that preceded treatment compared to the post-treatment estrus. In summary, low P(4) during diestrus was associated with smaller LS, reduced LBF, and larger FS in the treatment cycle, but not in the subsequent cycle.

Plasma progesterone concentrations in the mid-luteal phase are dependent on luteal size, but independent of luteal blood flow and gene expression in lactating dairy cows.

The objective of the present study was to investigate if plasma progesterone (pP(4)) concentrations are dependent on luteal size, blood flow, or gene expression in luteal tissue. To induce cycles with high and low pP(4) concentrations, respectively, 20 lactating dairy cows received either a single treatment with 25 mg prostaglandin F(2α) (PGF(2α)) on Day 4 Hour 12 (PG1; n=8), or two treatments (25 mg PGF(2α) each) on Day 4 Hours 0 and 12 (PG2; n=12) of the estrous cycle (Day 1, Hour 0=ovulation). In four cows, ovulation occurred between 4 and 6d after the second PGF(2α) treatment; these cows and one lame cow were excluded from the study. In the 15 remaining cows with physiological interovulatory intervals, pP(4), area (LTA) and volume (LTV) of luteal tissue, as well as absolute (LBF) and relative (rLBF) luteal blood flow were determined on Day 9, and relative luteal P(4) (rLP(4)) as well as luteal mRNA expression of important receptors, angiogenic, vasoactive, and steroidogenic factors were quantified on Day 11 (±1) during two successive estrous cycles. Furthermore, rLP(4) was multiplied by LTV to produce a semiquantitative assessment of absolute luteal P(4) (LP(4)). There was no effect (P>0.05) of treatment (one or two PGF(2α) treatments), neither on pP(4) concentrations nor on any other parameter in the present study. Nevertheless, there was a lower LP(4) (P=0.01), LTA (P=0.03), and LTV (P=0.02), as well as tendencies of lower pP(4) (P=0.06) and LBF (P=0.09) at first compared with second diestrus. Plasma P(4) was related with LP(4) (r=0.43, P=0.04), LTA (r=0.65, P=0.0001), and LTV (r=0.43, P=0.02), but not with rLBF (r=-0.18, P=0.34). Furthermore, there was no significant correlation between gene expression of important steroidogenic factors and P(4) concentrations in luteal tissue. Results indicate that plasma P(4) concentrations in the mid-luteal phase were dependent on luteal size, but independent of blood flow and gene expression per luteal tissue unit.

Effects of induction of ovulation with GnRH or HCG on follicular and luteal blood flow in Holstein-Friesian heifers.

The objective of this study was to compare the effects of gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) on follicular blood flow (FBF) during the pre-ovulatory period and on luteal blood flow (LBF) during dioestrus in Holstein-Friesian heifers. Twelve animals were randomly divided into two groups and treated with either intramuscular injection of 100 µg GnRH or intravenous (IV) injection of 5000 IU hCG on Day 0 (oestrus, 48 h after administration of PGF(2α) ) to induce ovulation. Follicular size (FS), FBF and time of ovulation were recorded with colour Doppler sonography at 0, 1, 3, 6, 12 and 24 h after GnRH and hCG treatment. Luteal size (LS) and LBF were investigated on Day 9 and 12 after ovulation. Plasma samples were taken to determine total oestrogens (E(tot) ) and progesterone (P(4) ) after each examination. Ovulation occurred between 24 and 48 h after treatment in all animals. No difference (p > 0.05) was observed in FS between the two treatment groups. Follicular blood flow was higher in the hCG group than that was in GnRH group at 1 h after treatment (p < 0.01). Total oestrogens were also higher (p < 0.01) in the hCG group than GnRH group; however, this difference was only obvious at 12 h after treatment. No difference (p > 0.05) was observed in LS, LBF or P(4) levels on Day 9 and 12 between treatment groups. In conclusion, the results suggest that induction of ovulation with hCG and GnRH has a temporary effect on FBF and oestrogen levels while no effect on the size of corpora lutea, LBF and P(4) levels was observed.

Luteal blood flow increases during the first three weeks of pregnancy in lactating dairy cows.

The objectives of this experiment were to characterize luteal blood flow in pregnant and non-pregnant cows and to determine its value for early pregnancy diagnosis. Lactating dairy cows (n = 54), 5.2 ± 0.2 y old (mean ± SEM), average parity 2.4 ± 0.2, and ≥ 6 wk postpartum at the start of the study, were used. The corpus luteum (CL) was examined with transrectal color Doppler ultrasonography (10.0-MHz linear-array transducer) on Days 3, 6, 9, 11, 13, 15, 18, and 21 of the estrus cycle (estrus = Day 0). Artificially inseminated cows (n = 40) were retrospectively classified as pregnant (embryonic heartbeat on Day 25; n = 18), nonpregnant (interestrus interval 15 to 21 d, n = 18), or having an apparent early embryonic loss (interestrus interval >25 d, n = 4). There was a group by time interaction (P < 0.001) for luteal blood flow from Days 3 to 18; it was approximately 1.10 ± 0.08 cm(2) (mean ± SEM) on Day 3, and increased to approximately 2.00 ± 0.08 cm(2) on Day 13 (similar among groups). Thereafter, luteal blood flow was numerically (albeit not significantly) greater in pregnant cows, remained constant in those with apparent embryonic loss, and declined (not significantly) between Days 15 and 18 in nonpregnant cows. Luteal blood flow was greater in pregnant than in nonpregnant (P < 0.05) and nonbred cows (P < 0.05, n = 14) on Day 15 (2.50 ± 0.16, 2.01 ± 0.16, and 2.00 ± 0.18 cm(2), respectively) and on Day 18 (2.40 ± 0.19, 1.45 ± 0.19, and 0.95 ± 0.21 cm(2)). In cows with apparent early embryonic loss, luteal blood flow was 2.00 ± 0.34 and 2.05 ± 0.39 cm(2) on Days 15 and 18, which was less (not significantly) than in pregnant cows, but greater (P < 0.05) than in nonbred cows on Day 18. Although mean luteal blood flow was significantly greater in pregnant than nonpregnant (and nonbred) cows on Days 15 and 18, due to substantial variation among cows, it was not an appropriate diagnostic tool for pregnancy status.

Luteal blood flow is a more appropriate indicator for luteal function during the bovine estrous cycle than luteal size.

The objective of this study was to assess the reliability of luteal blood flow (LBF) as recorded by color Doppler sonography to monitor luteal function during the estrous cycle of dairy cows and to compare the results with that for the established criterion luteal size (LS) as determined by B-mode sonography. In total, 14 consecutive sonographic examinations were carried out in 10 synchronized lactating Holstein-Friesian cows (Bos taurus) on Days 4, 5, 6, 7, 8, 10, 12, 14, 16, -5, -4, -3, -2, -1 of the estrous cycle (Day 1=ovulation). Plasma progesterone concentrations in venous blood (P(4)) were quantified by enzyme immunoassay. Luteal size was determined by sonographic measurement of the maximal cross-sectional area of the corpus luteum (CL). Luteal blood supply was estimated by calculating the maximum colored area of the CL from power Doppler sonographic images. Luteal size doubled during the luteal growth phase (until Day 7) and remained at this level during the luteal static phase (Day 8 to 16) before decreasing rather slowly during luteal regression (Days -5 to -1). Luteal blood flow doubled during the growth phase, doubled furthermore during the static phase, and decreased rapidly during luteal regression. Thus, LBF values represented highly reliable predictors of luteal status. Luteal blood flow predicted reliably a P(4)>1.0 ng/mL by reaching only 35% of the maximal values, whereas LS had to exceed 60% of the maximal values to indicate reliably a functional CL. It is concluded that LBF reflected luteal function better than LS specifically during luteal regression.

Effects of human chorionic gonadotropin on luteal blood flow and progesterone secretion in cows and in vitro-microdialyzed corpora lutea.

To check human chorionic gonadotropin (hCG) effects on luteal blood flow (LBF) and progesterone (P(4)) synthesis, six cows received either 3000 IU hCG or saline (NaCl) on Day 7 (Day 1=ovulation) during two estrous cycles. Plasma P(4) and LBF were measured before (0h) and up to 48h after treatment. Luteal blood flow increased by 51% (P<0.05) at 1h after hCG administration and returned to baseline levels thereafter. Plasma P(4) levels were increased from pretreatment levels by 30% at 1h (P=0.05) and 81% at 48h (P=0.02) after hCG treatment. In contrast, NaCl did not cause changes in LBF and P(4) (P>0.05). Additionally, central and peripheral parts of 14 abattoir-derived corpora lutea of the mid-luteal phase (Day 8 to 12) were perfused with Ringer solution in an in vitro microdialysis system, supplemented with 50 or 150 IU/mL hCG for 1h. Application of 50 IU/mL hCG showed no influence on P(4) response (P>0.05) in both central and peripheral parts, whereas 150 IU/mL hCG resulted in an increase of P(4) synthesis (P=0.002) in the central parts only. In vivo, hCG provoked an immediate and long-term rise in P(4) but only a temporary elevation of LBF. Luteal blood flow itself does not seem to be the exclusive cause for an increase in P(4), because the in vitro data clearly showed direct effects of hCG on P(4) secretion. Interestingly, different P(4) secretion patterns could be found between central and peripheral parts of the corpus luteum in both control and hCG perfused corpora lutea.

Ductus venosus shunting in marmoset and baboon fetuses.

OBJECTIVES: The increased shunting of blood through the ductus venosus (DV) during stress situations is an important mechanism that ensures fetal survival. Although primate fetuses may serve to study the function of this important venous shunt, the rate of DV shunting has not been determined in non-human primates under normal conditions. METHODS: DV shunting was measured in 11 marmoset (eight mothers) and eight singleton baboon fetuses in mid and late gestation using Doppler ultrasound. RESULTS: DV shunting in marmosets was 39 +/- 20% (mean +/- SD) and 28 +/- 8% in baboon fetuses. Umbilical vein (UV) blood volume rate increased significantly in baboons with gestational age (GA) (UV flow volume = -111.8 + 1.6*GA; r = 0.94, P < 0.05) and slightly in marmosets (UV flow volume = -10.37 + 0.13*GA; r = 0.42, P > 0.05). Both UV diameter (r = 0.84) and the time-averaged mean UV flow velocity in baboons depended on GA (r = 0.8, P < 0.05). Distinct pulsation was found in marmoset fetuses in the UV (pulsatility index (PI) = 1.3 +/- 0.9) and the DV (PI = 1.9 +/- 1.2) with zero blood flow velocity during atrial contraction. CONCLUSIONS: A high level of pulsation is observed in the UV in marmoset fetuses. DV shunting in marmosets is higher than in baboon fetuses.